The entorhinal cortex (EC), main input structure to the hippocampus, gets innervated by serotonergic terminals from the raphe nuclei and expresses 5-HT-receptors at high density. Using extra- and intracellular recording techniques we here investigated the effects of serotonin on population and cellular responses within the EC.Stimulation in the lateral entorhinal cortex resulted in complex field potential responses in the superficial EC. The potentials are composed of an early antidromic and a late orthodromic component reflecting the efferent and afferent circuitry.Serotonin (5-HT) reduced synaptic potentials of the stimulus evoked extracellular field potential at all concentrations tested (0.1–100 μM; 59%-depression by 10 μM serotonin), while the antidromic response was not significantly changed by up to 50 μM 5-HT. Depression of field potential responses by serotonin was associated with a significant increase in paired-pulse facilitation from 1.15 to 1.88.The effects of serotonin on field potential responses were mimicked by 5-HT1A-receptor agonists (8-OH-DPAT, 5-CT) and partially prevented by the 5-HT1A-receptor antagonist (S-UH-301). Moreover, the 5-HT1A-receptor antagonist WAY100635 reduced the effect of 5-CT.Fenfluramine, a serotonin releaser, mimics the effects of serotonin on stimulus-evoked field potential responses, indicating that synaptically released serotonin can produce the changes in reactivity to afferent stimulation.Depression of isolated AMPA-receptor mediated EPSCs by serotonin as well as fenfluramine was associated with an increase in paired pulse facilitation, indicating a presynaptic locus of action.We conclude that physiological concentrations of serotonin potently suppresses excitatory synaptic transmission in the superficial entorhinal cortex by a presynaptic mechanism.